Cohesin is a multi-subunit protein complex that orchestrates proper segregation of chromosomes by mediating cohesion between sister chromatids. Defects in the cohesion pathway lead to certain developmental diseases, as well as chromosome segregation defects like those in cancer. Cohesin binds centromeres where it helps mount chromatids onto spindle microtubules. The complex also binds discrete sites in relation to the transcriptional landscape of the genome and recent work suggests that cohesin plays significant roles in transcriptional regulation. In the model eukaryote yeast Saccharomyces cerevisiae, cohesin binding is dynamic in euchromatic domains where genes are poised for transcription. The complex is also heavily enriched at heterochromatic domains where transcription is suppressed. These domains functionally resemble heterochromatin of higher eukaryotes and have been useful in deciphering principles of regional inactivation and epigenetic control. The Gartenberg laboratory has used the yeast system to define principles of chromosome architecture as they relate to chromosome function. Using site-specific recombination and other novel molecular genetic strategies, the laboratory has focused on how yeast heterochromatin intersects with the sister chromatid cohesion pathway. The laboratory recently discovered that Sir2, the evolutionarily conserved protein deacetylase responsible for yeast heterochromatin assembly, retains cohesin at heterochromatic loci. Aim 1 investigates the molecular basis of this event and the functional consequences of cohesin on heterochromatic silencing and genome stability. Other transcriptional regulators also appear to direct cohesin to unexpressed genes. In aim 2, the tools developed for the study heterochromatic cohesion will be used to determine how cohesin arrives at non-heterochromatic genes and how the complex is utilized when transcription is activated. One particular chromosomal domain under study contains heterochromatin juxtaposed to an active tRNA gene. Previously the Gartenberg laboratory found that the tRNA gene is required for heterochromatic cohesion. Now the laboratory has learned that the domain localizes with nuclear pores in a cohesin-dependent manner. In aim 3, the molecular basis for this new dimension in higher order chromosomal organization will be investigated. PUBLIC HEALTH RELEVANCE: Human diseases caused by mutations in the cohesin pathway (cohesinopathies) display a host of transcriptional and/or heterochromatin structure defects. To understand what goes wrong in these diseases, we need to first fully account for how cohesin normally functions at transcriptionally repressed and activated genes. Using budding yeast as a model system, we will investigate the behavior of cohesin on euchromatic genes and how the complex influences the silencing, structure and stability of heterochromatic loci.